In order to establish a base of structural knowledge concerning structure, function, and regulation in the enigmatic family of manganese metalloenzymes, we have selected the metallohydrolase rat liver arginase as the paradigm for protein engineering and rational ligand design experiments. This enzyme is extraordinary in that it contains a binuclear, spin-coupled manganese cluster in its active site that is implicated in the chemistry of ariginine hydrolysis. In addition to yielding the first structure of a mammalian urea cycle enzyme (where cytosolic arginase catalyzes the hydrolysis of arginine into omithine plus urea), our structural studies of arginase complement studies of its role in nitric oxide (NO) biology. The activities of nitric oxide synthase and arginase in various tissues are reciprocally coordinated in order to modulate NO-dependent processes. In this role, we have implicated arginase as a potential target for therapeutic intervention in the treatment of erectile dysfunction. We have achieved key goals outlined in the original grant proposal, and we now request continued support for X-ray crystallographic studies of arginase, its site-specific variants, and its inhibitor complexes. Additionally, building on our successful design and evaluation of potent boromc acid-based arginase inhibitors in the current funding period, we request support for exploring the development of new inhibitors in a structure-based design approach. New inhibitor designs may exhibit enhanced properties such as affinity and membrane permeability, which may facilitate physiological studies of arginase inhibition in the laboratories of our collaborators.